Genetic ancestry shapes dengue virus infection in human skin explants

Abstract

Dengue is the most prevalent arthropod-borne viral disease of humans, with over half the world's population at risk. Infection with any of the four dengue virus (DENV) serotypes is most often self-limiting, but a significant number of cases present with severe dengue characterized by vascular leakage that may be fatal. African ancestry is associated with protection against severe dengue, but the mechanisms are unknown. Using skin explants from genetically defined donors, we show that European ancestry skin has a much stronger inflammatory response to DENV than African ancestry skin, eliciting markedly increased infiltration, infection and migration of resident Langerhans cells, macrophages, and dendritic cells. The effect was seen with all dengue serotypes and Zika virus and in the presence of heterotypic immune serum. Genetic pathways associated with inflammation, interferon (IFN)-α, and inflammatory cytokine signaling were enriched in European relative to African ancestry skin following infection. Infiltration and infection of macrophages in African ancestry skin increased to that of European skin after blocking IFN-α and providing interleukin-1β. Polymorphic variants in RXRA, OAS1-3, and TGFB1 genes that were more frequent in European donors were associated with increased virus replication. Paradoxically, European ancestry skin cells had increased expression of OAS3 in response to virus and type I IFN. Thus, the limited inflammatory response of African ancestry skin to infection restricts replication and spread of dengue and other flaviviruses. Genetic ancestry should be considered when predicting a patient's likelihood of severe dengue, and when assessing efficacy and adverse events associated with dengue vaccines.

Keywords: ancestry; dengue virus; inflammation.

Fig. 1.

Defining genetic ancestry and cellular responses to DENV infection. (A) Biogeographical ancestry of all skin donors showing the proportion of African, European, Asian, and Native American ancestry based on ADMIXTURE analysis. (B) Principal component analysis of biogeographical ancestry overlaid on 809 reference samples for population structure. (C) Association between NS3-expressing cells in epidermis and dermis as a percentage of the total imaged area and the proportion of European ancestry (n = 38). R-squared and P value were determined by Pearson correlation and simple linear regression. (D and E) Representative images of immunofluorescence of skin sections from EA and AA individuals 24 h after DENV-2 inoculation stained with antibodies to viral NS3 (D) and the indicated cell markers (E). Dotted lines outline the epidermal–dermal junction. (Scale bar, 100 µm.) Blue staining represents nuclei, green represents viral NS3, and magenta represents cell-specific markers. (F) Quantification of AE1⁺ keratinocytes in uninfected (UI), mock-infected and DENV-infected skin (Left) and of AE1⁺NS3⁺ keratinocytes in infected skin (middle) in AA (n = 15) and EA (n = 21) specimens at 24 h. The relationship between infected and total keratinocytes as a function of European ancestry and the ratio of cells in the infected versus mock condition (n = 38) is shown on the Right. (G–I) Similar analyses as (F) shown for (G) CD207⁺ Langerhans cells in the epidermis in a subset of individuals (AA = 10, EA = 8) and (H) CD1c⁺ DC and (I) CD163⁺ macrophages in the dermis (AA = 15, EA = 21). (J) The number of migrated cells in media normalized to skin area from AA (n = 4) and EA (n = 4) skin after mock and DENV-2 infection at 24 and 48 h. (K) Association between number of migrated cells and DENV genomes expressed as FFU equivalents/mL, AA (n = 4) and EA (n = 4). P and r values were determined by simple linear regression. (L) Quantification of NS3⁺ cells in epidermis and dermis in AA (n = 4) and EA (n = 4) skin 24 h after infection with each of the four serotypes of DENV and Zika virus. (M) Quantification of NS3⁺ cells in epidermis and dermis and of NS3⁺ macrophages 24 h after DENV-2 infection in the presence of naïve or pooled anti-DENV-3 serum in AA (n = 4) and EA (n = 4) skin. The horizontal lines in all graphs represent medians. Between group analyses were done by the unpaired t test and within-group analyses were done by one-way ANOVA followed by Tukey’s multiple comparisons.

Fig. 2.

Ancestry-related differences in transcriptomic and inflammatory responses. (A) The number of differentially expressed genes that were increased or decreased with European ancestry at different false discovery rate cutoffs at 24 h post infection. (B) Gene ontology network for genes with positive interaction effects showing pathways enriched with European ancestry following DENV infection. All significant pathways (FDR < 0.05) are shown. (C) Bubble plot showing gene pathways enriched following DENV infection and their relationship to European ancestry. Gene ontology enrichment analyses were performed separately for genes with a stronger or decreased response to infection (log2 fold-change greater than two in each direction of the effect). Only pathways with enrichment at FDR < 0.05 and at least four terms per cluster are shown. (D) Gene ontology terms within inflammatory response pathways and their relationship to European ancestry. (E) Gene set enrichment analysis of pathways associated with inflammatory and antiviral responses (Left) and with cytokine signaling (Right) that are enriched in EA skin relative to AA skin following DENV infection at 24 h. (F) Immunofluorescence of skin sections from EA and AA individuals 24 h after DENV-2 inoculation stained with antibody to IL-1β. (Scale bar, 100 µm.) Blue staining represents nuclei, and white staining represents IL-1β. (G) Quantification of IL-1β staining as a percentage of total imaged area at intervals after mock and DENV infection in skin from AA (n = 3) and EA (n = 5) donors. Shown are means and SE of the mean. Analysis was done by the Mann–Whitney U test comparing DENV conditions between AA and EA skin at each time point. (H) Quantification of CD163⁺ macrophages (Left) and CD163⁺NS3⁺ macrophages (Right) in AA (n = 3) and EA (n = 4) skin 24 h after mock or DENV-2 infection in the presence of IL-1β with and without anti-IFN-α, or anti-IL-1β with and without IFN-α, or isotype control antibody delivered by microneedle array. Horizontal lines represent medians. Analysis was done by one-way ANOVA followed by the Tukey test.

Fig. 3.

Genetic polymorphisms and ancestry-related differences in DENV infection. (A) Relationship between European ancestry and NS3⁺ staining in epidermis and dermis 24 h after DENV-2 infection for 60 SNPs. The x-axis shows the P values obtained when comparing the median levels of European ancestry between the genotypes for each SNP. The y-axis shows the P values obtained when comparing the median levels of DENV-2 infection between the genotypes for each SNP. Dashed lines show the threshold for significance based on a Benjamini–Hochberg correction using a false discovery rate of 0.1. Polymorphisms that are significantly associated with both ancestry and infection are shown in red. (B) Allele frequency for the six significant SNPs in the cohort of AA (n = 24) and EA (n = 35) donors. (C) Genotypes and/or haplotypes of AA (n = 15) and EA (n = 21) donors for SNPs that are significantly associated with both ancestry and infection in epidermis at 24 h. (D) Flow cytometry dot plots of epidermal cells from an AA and EA donor stained with and without 2H2 antibody to prM protein 24 h after mock or DENV-2 infection of cell suspensions. (E) Association between the percent infection of epidermal cells and the proportion of European ancestry of donors (n = 13). R-squared and P values were determined by Pearson correlation and simple linear regression. (F) DENV genomes expressed as FFU/mL (Left) and mRNA expression of OAS1, OAS2, and OAS3 in epidermal cells 24 h after mock or DENV-2 infection relative to baseline (control 0 h) (Right) in AA (n = 4) and EA (n = 5) individuals. (G) Relative expression of OAS1, OAS2 and OAS3 mRNA in epidermal cells 18 h after control or 2,000 U/mL recombinant IFN-α stimulation in AA (n = 5) and EA (n = 7) individuals. Horizontal lines represent medians. Within-group analyses were done using the Wilcoxon matched pairs signed rank test and between-group analyses were done using a Mann–Whitney U test.